Procedure for enzymatic production of semi-synthetic penicillins

ABSTRACT

A process for the enzymatic hydrolysing of diacyl-penicillins to semi-synthetic penicillins of the general formula:   The hydrolysis takes place in the presence of the enzyme penicillin-acylase produced by microorganisms.

Witale et all,

[ PROCEDURE FOR ENZYMATIC PRODUCTION OF SEMI-SYNTHETIC PENICILLINS [75]Inventors: Eupremio Vitale; Guido Guerra,

both of Bologna, Italy [73] Assignee: Alfa lFarmaceutici S.p.A.,Bologna,

Italy [22] Filed: Nov. 10, 1971 [21] Appl. No.: 197,513

[30] Foreign Application Priority Data Nov. 7, 1970 Italy 3608 -A/70[52] US. Cl. 195/29 [51] Int. Cl C12d 9/00 [58] Field of Search 195/36P, 29

[56] References Cited UNITED STATES PATENTS 3,239,427 3/1966 Huang etal. 195/36 P Oct. 16, 1973 Primary Examiner-Alvin E. TanenholtzAttorney-Richard K. Stevens et al.

[57] ABSTRACT A process for the enzymatic hydrolysing ofdiacylpenicillins to semi-synthetic penicillins of the general formula:

s 2011; n-oo-Nrr-on-ofi 0-011,

The hydrolysis takes place in the presence of the en zymepenicillin-acylase produced by microorganisms.

11 Claims, No Drawings 1 PROCEDURE lFOR ENZYMATIC PRODUCTION OFSEMI-SYNTHETIC PENllCllLLINS The present invention is concerned with anew enzymatic procedure for the preparation of semi-syntheticpenicillins. More specifically, it refers to a new enzymatic procedureto obtain semi-synthetic penicillins with the general formula (I):

where R represents a group chosen among a-aminobenzyl, a-car-boxybenzyl,2 or 3 thienylmethyl, 2 or 3 thienylaminomethyl, or an isoxazole groupwith the general formula:

(in which R, indicates a phenyl group or a 2- chlorophenyl or 2,6dichlorophenyl); R can also represent a group with the formula:

""RB TT R.

in which I1 meiosis aryl, afyl oi l, arylrn'e'rcaptyl possiblysubstituted by one or more halogen atoms); R indicates a hydrogen or a(C C alkyl group, (C -C alkoxyl, amino, (C -C monoalkyl-amino, (C -Cdialkyl-amino, carboxyl, (C -C carboxyalkyl, carboxymethyl acetoxyl,carboxybenzyl, and, as a special case, R and R together can belong to aC -C cycloaliphatic ring;

R represents hydrogen or, in the special case where R and R togetherform a C,C cycloaliphatic ring, R represents an amino, (C -Cmonoalkylamino, or (C -C dialkylamino group.

The present invention refers also to the preparation of non-toxic saltsand esters of semi-synthetic penicillins with formula (I). Suchnon-toxic salts include those of sodium, potassium, calcium, magnesium,aluminum, ammonium and substituted ammonium, such as those formed byreaction with non-toxic amines, e.g., triethylamine, procaine,dibenzylamine, N-benzyl-B- phenylethylamine,N,N'-dibenzylethylendiamine, N-

alkylpiperidin, and other amines which are usually employed to formsalts with benzyl penicillin.

Among the non-toxic esters of semi-synthetic penicillins with formula(I) can be quoted: (C -C alkyl, (C -C acyloxymethyl, p-methoxybenzyl,pnitrobenzyl, benzyhydryl, phenacyl; p-bromophenacyl, trimethylsilyl,benzyl.

More specifically, the present invention concerns an enzymatic procedurefor the preparation of semisynthetic penicillins, specified above informula (I), (or of their non-toxic salts or esters) characterized bythe fact that said procedure includes the enzymatic dephenylacetylationof diacylpenicillins with the general formula (II):

42C, at an alkaline pH, preferably between pH 7.1 and 8.5.

According to the present invention, the enzymati; dephenylacetylation ofdiacylpenicillins with formula (II) occurs by action of the enzymepenicillin-acylase, produced by several microorganisms. The preparationof the semi-synthetic penicillins represented in formula (I) has beendoneup to now by chemical acylation of 6-amino-penicillanic acid.Different reactive derivatives of carboxylic acids were used for thisreaction, such as acid chlorides, anhydrides or mixed anhydrides, oranhydrides coming from the corresponding free carboxylic acid and6-amino-penicillanic acid in the presence ofa condensing agent, as forinstance dicyclohexylcarbodiimide. The 6-amino-penicillanic acid to beused was prepared by means of microciological hydrolysis ofbenzylpenicillin. These procedures are estensively described inscientific literature.

7 "iii th e Belgian Pat. No.73 8.ll0 a pr ocedure is de- Nevertheless,this procedure is subject to serious limitations. In fact it can beapplied only to the preparation of isoxazolyl penicillins, in so far asthe dephenylacetylation by chemical reaction with primary amines isfeasible only in these limited cases due to steric hindrances of theisoxazole residue. Furthermore, during chemical dephenylacetylation, aconsiderable amount of epimerization is observed at position 6 in thepenicillin molecule, with a resulting partial loss of antimicrobicactivity. For such reason, other semi-synthetic penicillins oftherapeutic or commercial interest cannot be prepared with this process.

the object of the present invention, that the diacylpenicillins definedin formula (II) can easily be dephenylacetylated in high yields, withoutcausing epimerization in position 6 of the penicillin nucleus, byenzymatic hydrolysis using the enzyme penicillin-acylase, which ishighly specific for the phenacetyl group. Besides the fact that thepresent invention is particularly versatile, allowing the preparation ofa great number of semi-synthetic penicillins, it has the advantage thatby using mild treatments such as enzymatic dephenylacetylation, it doesnot produce epimerization phenomena of the hydrogen linked to the carbonatom in position 6 in the penicillinic structure of the molecule, as isoften the case with chemical methods. The object of the presentinvention is, therefore, an enzymatic procedure for semi-syntheticpenicillins (and their non-toxic salts and esters) defined above in thegeneral formula (I). Said procedure is characterized by the fact thatthe dephenyl-acetylation of the diacylpenicillins specified in formula(II) is carried out by enzymatic hydrolysis in the presence of theenzyme penicillin-acylase, produced by microorganisms, at a temperaturebetween C and 50C, preferably between C and 42C, and at an akaline pH,preferably between pH 7.1 and 8.5. The preparation of semisyntheticpenicillins, according to the present invention, occurs in the presenceof penicillin-acylase or in the presence of enzymatic preparations fromany source containing such enzyme.

For the enzymatic preparation of semi-synthetic penicillins defined informula (I), microorganisms producing penicillin-acylase can be used,chosen among those capable of attacking the amide group in position 6 ofthe benzylpenicillin molecule, with the formation of 6-amino-penicillanic acid. This property can be evidenced through the factthat such microorganisms are able to inactivate penicillin G to theextent of at least 20 percent within 24 hours at 38C, giving as a resulta solution in which the penicillin G can be reactivated at leastpartially by addition of phenacetyl chloride. These microorganismsinclude molds, yeasts and bacteria and can be chosen among the followinggenera: Alternatia, Aspergillus, Botritis, Cephalosporium, Cryptococcus,Emericellopsis, Eipcoccum, Epiolermophyton, Fusarium, Mucor, Penicillum,Phoma, Trichoderma, Trichophyton, Trichosporon, Streptomyces,Aerobacter, Alcaligenes, Bordetella, Cellulomonas, Corynebacterium,Erwinia, Escherichia, Flavobacterium, Micrococcus, Proteus, Pseudomonas,Salmonella, Sarcina, Xantomonas, Torulopsis, Rhodotorula, Arthrobacter.

These microorganisms can be used for the enzymatic synthesis ofpenicillin, as in formula (I), according to the present invention eitherunder the form of cells collected from culture media or as acetonicpowders, or as cells adsorbed on an inert carrier or, finally, in theform of cellular extracts (enzymatic preparations). In the scope of thepresent invention it is advantageous, but not essential, to use assource of enzymes specially selected microbic strains, which bymutations and successive passages have been made resistant to highconcentrations of phenylacetic acid (l,0003,000'y/ml), and are thus ableto produce large amounts of the enzyme penicillin-acylase.

Therefore, the enzymatic procedure for the preparation of penicillinsspecified in formula (I) consists in the dephenylacetylation ofdiacylpenicillins as in formula (II) by enzymatic hydrolysis in thepresence of penicillin-acylase producing microorganisms, or of anenzymatic preparation containing penicillinacylase, at a temperaturecompatible with the stability of both the enzymatic system and thepenicillin molecule. Such temperature includes the interval between 20Cand 50C, preferably between 30C and 42C.

As any enzymatic process, the enzymatic dephenylacetylation ofdiacylpenicillins such as formula (II) is sensitive to variations of pH.To obtain scmisynthetic penicillins according to the present inventionthe dephenylacetylation is performed in the pH interval between 7.1 and8.5, preferably between 7.5 and 8.0.

The quantity of diacylpenicillin as in formula (II) which can bedephenylacetylated by a given amount of enzymatic material within a timelapse compatible with both industrial requirements and stability of thepenicillin molecule depends on the enzymatic activity present in thebiological material to be used. Therefore, it is advantageous todetermine, a priori; such activity based on the capacity ofpenicillin-acylase to hydrolize penicillin G into 6-amino-penicillanicacid and phenylacetic acid. For successful execution of the proceduredescribed in the present invention, for each mole of diacylpenicillin asin formula (II) one shall use that amount of biological material capableof splitting into 6-amino-penicillanic acid a mole of benzylpenicillin,at 37C and at a constant pH of 7.8, in a 3 percent solution in 10 hoursup to 65 percent of the theoretical.

At the end of the enzymatic reaction, the semisynthetic penicillinformed can be isolated from the solution by means of different knowntechniques. A widely used method consists in the removal of the cellularor enzymatic material by filtration or centrifugation. The penicillincan be extracted from the clear solution obtained by means of an organicsolvent, insoluble in water, after acidification with a strong acid. Thepenicillin can then be precipitated from the organic phase in the formof sodium salt by adding a saturated organic solution ofsodium-2-ethyl-hexanoate. The precipitate is collected, by filtration,washed with a suitable solvent, and dried under vacuum.

The product so obtained is, in general, sufficiently pure for practicalpurposes. Alternatively, the penicillin can be precipitated in the formof a crystalline salt poorly soluble in water by adding a solution of asubstance with which the penicillin forms the required insoluble salt.To this purpose, the salts of N,N'-dibenzylethylendiamine,dihydroabietylamine, procaine, N- ethylpiperidine, etc., can beadvantageously used. The details for the performance of the procedurescovered by the present invention are further illustrated by thefollowing examples, which are not to be taken as restrictive in anyrespect.

EXAMPLE 1 Production of Escherichia coli penicillin-acylase producingcells.

Cells of a strain of Escherichia coli (ATCC 9637), previously maderesistant to 2,0007/ml of phenylacetic acid, are cultivated at atemperature of 30C on a medium of the following composition:

Corn steep liquor 2.8% Peptone 0.1% Casein hydrolisate l.0% Na,HPO, 2H,0l.8% K,HPO. 1.8% Vaseline oil 0.02% Na-phenylacetate 0.2% pH 6.8-7.0

in a laboratory fermenter under agitation (650 rpm.) and spargeraeration (6 liters per hour/liter) until the optical density of themedium reaches the value of 99/100 at the wavelength of 610 my, read ona colorimeter.

The growth normally takes 8-9 hours. After this time, the medium iscentrifuged and the harvested cells are dispersed in an equal volume ofsaline. This suspension contains 5.5 X 10 cells per ml. To determine theenzymatic activity of the cellular suspension obtained as above, 10 mlof this suspension are incubated at 37C and ph 7.8 with differentamounts of sodium salt of benzylpenicillin. At regular time intervals,samples of the mixture are analyzed for the quantity of benzylpenicillinremaining intact. The results obtained are reported in the followingtable:

Time of incubation Quantity of benzylpenicillin As one can see, thetransformation of about 65 percent in 10 hours is obtained with thesolution which originally contained 30 mg/ml of benzylpenicillin.

EXAMPLE 2 Production of an enzymatic solution of penicillinacylase fromEscherichia coli.

Cells of a strain of Escherichia coli (ATCC 9637), made resistant to2,000'y/ml of phenylacetic acid,are cultivated as described underExample 1. The harvested and washed cells are suspended in distilledwater to a concentration of 10 X 10 cells/ml. The dispersion so obtainedis layered with 3 percent (vol/vol) of methylisobutylketone, and themixture is kept at 38C for 6 hours. The cellular material is separatedby centrifugation. The clear solution obtained shows an enzymaticactivity capable of decomposing 65 percent of 43 mg/ml ofbenzylpenicillin in 10 hours at 38C and at pH 7.8.

EXAMPLE 3 Procedure for the preparation of diacylpenicillins.

The procedure described in this example is not claimed under the presentinvention, but is only used to illustrate the method of preparation ofdiacylpenicillins, used as starting material for the preparation ofsemi-synthetic penicillins according to the present invention.

A. Preparation of the p-nitrobenzyl ester of benzylpeni cillin (compoundA).

To a mixture of 74.5 g of potassium benzylpenicillin plus 500 ml ofdimethylformamide are added 39 g of p-nitrobenzyl bromide. The mixtureis kept at 5560C for hours under agitation. The mixture is allowed tocool; is diluted with 300 ml of benzene, and is then poured into 500 mlof water. The organic phase is separated and washed three times withwater to eliminate the benzylpenicillin which has not reacted. Thebenzene solution, dried by means of anhydrous Na SO will be useddirectly without isolating the compound obtained. The analysis shows acontent of benzylpenicillin ester corresponding to 70.8 g (yield: 75percent). B. Preparation of the acetoxymethy] ester of benzylpenicillin(compound B).

A mixture of 45.6 g of N-ethylpiperdine salt of benzyl-penicillin and 79ml of bromomethylacetate in 200 ml of dimethylformamide is kept at roomtemperature for 16 hours under stirring. It is then diluted with 500 mlof chloroform and poured into water. The organic phase is washedrepeatedly with water to eliminate any trace of unreactedbenzylpenicillin. The washed solution is finally dried on anhydrous NaSO and is used directly in the synthesis of diacylpenicillins. Theorganic solution obtained contains 33.6 g of the acetoxymethyl ester(yield: 8.1 percent).

C. Preparation of trimethylsilyl ester of benzylpenicillin (compound C).

74.5 g of potassium benzylpenicillin are dispersed in 750 ml ofchloroform, and then, gradually, 32.6 ml of trimethylchlorosilane areadded. During this addition the temperature must not rise above 26C.After the addition, the mixture is kept at 25-27C for one hour underconstant agitation. After filtration, the filtrate is utilized directlyin the successive steps to prepare diacylpenicillins. The organicsolution contains 79.6 g of trimethylsilyl ester of benzylpenicillin(yield: 96 percent).

D. General procedure for the preparation of iminochlorides of protectedbenzylpenicillin.

The organic solution containing 0.1 mole of protected benzyl-penicillin,prepared according to one of the points A,B,C is treated with 0.12 molesof quinoline and is cooled externally to-35C. Under agitation, 0.1 moleof finely powdered PCl is added, and the mixture is kept at 30 to 35Cfor 3 hours, under constant agitation and protection from humidity. Atthe end of the reaction, the mixture is filtered from the quinoline HCl,the solvent is evaporated under reduced pressure, and the residue isrepeatedly distilled under vacuum with anhydrous benzene at atemperature not above 25C in order to remove the phosphorus oxychlorideformed in the reaction. The residue, free from phosphorus oxychloride,is dissolved in 200 ml of anhydrous benzene and utilized directly forthe preparation of protected diacylpenicillins. The yield is practicallyquantitative. E. General procedure for the preparation of protecteddiacyl-penicillins.

The benzene solution obtained under D containing the iminochloride ofthe esterified benzylpenicillin, is treated with finely powdered sodiumsalt of the required carboxylic acid (0.15 moles). The mixture is keptunder agitation at room temperature for a period of 20-24 hours. At theend of the reaction, the mixture is filtered from the excess of sodiumsalt and from the sodium chloride formed. The filtrate is washedrepeatedly with water and with a 3 percent sodium bicarbonate solution(when trimethylchlorosilane is used for the protection of thebenzylpenicillin, the washing procedure is modified and a 5 percent HClsolution used for the final wash. During this aqueous-acid washing, thetrimethylsilyl ester of diacylpenicillin is hydrolized and will be foundas free acid in the final benzene solution, from which it can beisolated as sodium salt by treatment with Na-Z-ethylhexanoate.) Byevaporation of the solution under reduced pressure, the raw protecteddiacyl-penicillin is obtained.

F. General procedure for the preparation of diacylpenicillins free fromprotecting groups.

1. Removal of the p-nitro benzyl group.

The removal of a p-nitro group can be achieved through several methodsdescribed in scientific literature, all based on cathalytic reduction.One can, for instance, proceed as follows: the residue obtained as underpoint E after evaporation of the solvent, is dissolved in 200 ml ofethyl acetate. The solution obtained is hydrogenated on 5 percentcharcoal at atmospheric pressure in the presence of 10-14 ml oftriethylamine,

and 30 g of palladium catalyst. At the end of the reduction, thecatalyst is removed and the solution is repeatedly extracted with waterat pH 7.1-7.5. The pooled aqueous phases give by lyophylization thetriethylamine salt of the desired diacylpenicillin.

2. Removal of the p-bromophenacyl protecting group. The residue obtainedunder point E after evaporation of the solvent, which consists of thep-bromophenacylic ester of diacylpenicillin, is dissolved in 150 ml ofdimethylformamide. For 0.1 mole of initial product, 0.2 moles of sodiumthiophenolate are added. The mixture is kept under agitation at roomtemperature for 30 minutes and then diluted with acetone. The raw sodiumsalt of the desired diacylpenicillin is precipitated and collected byfiltration.

3. Removal of the acetoxymethyl protecting group. The residue obtainedunder point E is dissolved in ethyl ether and the resulting solution iskept under agitation at C with a percent HCl solution for half an hour.The organic phase is separated, washed with water, dried over Na SO andtreated with an equivalent amount of molar solution of sodium2-ethylhexanoate in methylisobutylketone. The sodium salt of thediacylpenicillin desired is obtained as a precipitate.

EXAMPLE 4 Preparation of sodium salts of diacylpenicillins.

The preparations described under this example are not claimed under thepresent invention. Diacylpenicillins are prepared according to thegeneral method described under Example 5. In the following table thedata referring to the different preparations are reported:

N-OH-CH O R-CO CH3 OON CHCOONa Com pound Protec- Percent number R tionyield 1 m M PBF V 44 N C-CH:

2 TMCS 71 f ii 1J1 CCH3 C1 CC II II N C-CH3 C1 4 TMCS 63 l NH: form D'lAlllllifionl.lnnud om pound I'rntnclvrcunl. number It Lion" yield 5 5.i i U in tial; 67

NH; form D 5 BMA 72 COONa 6 TMCS 77 CO0CHz- V W Vim 'IMCS 76.

TMCS 76 i T os 67 9 ([)CII3 M OCHa 10 (|)C2II5 PBF 53 C|2H- TMOS 66 4 tNH2 form D 12 if V 'IMCS 51 NH2 13 (I311- TMCS 63 I [NIH 55 S form I) 14TMCH 54 1 A NH, I V

P rotection=BMA=Bromomethylacetutc; PBF=p-bromopheimcyl bromide;TMCS=trimethylchlorosilanc; PN BB =p-nltrobcnzyl bromide.

EXAMPLE 5 Procedure for the preparation of Oxacillin.

53.6 g of sodium salt of N-(3-phenyl-5-methylisoxazolyl-4)benzylpenicillin (compound 1 of Example 4) are added to 1,000 ml of acell suspension of Escherichia coli prepared as under Example 1. The pHis adjusted to 7.8 and the mixture is kept under agitation and moderateaeration at 36-39C for 12-15 hours. The pH of the mixture is constantlykept between 7.5 and 8.0 by adding a 5 percent soda solution. Duringthis period, in order to neutralize the phenylacetic acid formed, 74 mlof the soda solution are used (92 percent of theoretical). At the end ofthe enzymatic hydrolysis, the suspension is centrifuged and the clearsolution is cooled to 05C.

The solution is now treated, under agitation, first with 250 ml of etherand then with percent HCl to reach pH 2.0. The organic phase isseparated and washed three times with 100 ml of water, and dried over NaSO The anhydrous ether solution is then treated with 100 ml of 1.0 molarsodium 2-ethylhexanoate in methylisobutylketone. The sodium salt ofOxacillin which precipitates is filtered and washed first with ether andthen with dry isopropanol. After drying under vacuum, 39.1 g of productare obtained showing a microbiological titer of 89 percent (yield289percent).

EXAMPLE 6 Procedure for the preparation of Chloxacillin. Using 7.5 g ofsodium salt of N-[3(2-chlorophenyl)-S-methylisoxazolyl-4]benzylpenicillin (compound 2under Example 4) and proceeding as described under Example 7, 41.6 g ofChloxacillin are obtained (yield: 90.6 percent) showing the followingcharacteristics:

Mlcrobiological titer 94% H O (Karl Fischer) 4.6%

lodometric titer 96% EXAMPLE 7 Procedure for the preparation ofDichloxacillin.

61 g of compound 3 as under Example 4 (sodium salt ofN[3(2,6-dichlorophenyl)S-methyl-isoxazolyl- 4]benzylpenicillin) are usedas described under Example 1. At the end of the enzymatic hydrolysis,the mixture centrifuged to remove the cellular material and theresulting clear solution is treated with one liter ofmethylisobutylketone. it is then acidified at 0C with 10 percent HCl toreach pH of 2 and the organic phase is separated, washed repeatedly withwater and dried over Na SO To the dry solution is added 120 ml of 1.0molar Na-Z-ethylhexanoate solution is methylisobutylketone and themixture is heated to 2528C under agitation. it is then kept at roomtemperature under constant agitation until it crystalizes completely.The precipitate is filtered and washed first with methylisobutylketoneand then with ether, and dried under vacuum. 44.2 g of Dichloxacillin(yield: 88 percent) are obtained, having the following characteristics:

Microbiological titer 91.3%

H,O (Karl Fischer) 4.2%

lodometric titer 95.1%

EXAMPLE 8 Procedure for the preparation of Ampicillin(D(-)aamino-benzyl-penicillin).

59 g of compound 4 of Example 4 are dissolved in 1 liter of enzymaticsolution of penicillin-acylase prepared as under Example 2. The pH isbrought to 7.8 and the solution is kept at 3639C for 9-12 hours. The pHis constantly kept between 7.5 and 8.0 with 5 percent soda. At the endof the enzymatic hydrolysis, the solution is cooled to 25C and acidifiedwith 10 percent HCl to pH 1.5-2.0. The acid solution is extracted threetimes with 100 ml of methylisobutylketone. The extracted aqueoussolution is concentrated under reduced pressure (2528C) after correctingthe pH to 7.0. The residue is filtered on a bed of filteraid mixed withactive charcoal. The solution obtained is brought to pH 4.84.9 underagitation and external cooling.

The Ampicillin which precipitates in the form of trihydrate is collectedby filtration and washed, first with a little ice water and then with 85percent (vol/vol) isopropanol. It is then dried in a warm air stream(40C). 41.6 g of Ampicillin trihydrate are obtained showing thefollowing characteristics:

Microbiological titer 89.6% H O (Karl Fischer) 12.8% Hydroxamic titer93.1% [11],, (c=0.25% in H 0 276 EXAMPLE 9 Procedure for the preparationof 6[()oz-amino-(2- thienyl)-acetamido] penicillanic acid.

Using 49.5 g of compound 11 as under Example 4 and proceeding asdescribed under Example 8, 30.6 g of 6[()a-amino(2-thienyl)-acetamido]penicillanic acid trihydrate are obtained (yield: percent) having thefollowing characteristics:

Hydroxamic titer 92% [01],, (c=0.5% in 0.1N HCl) +192 EXAMPLE 10Procedure for the preparation of 6[1-amino-l cyclohexyl-carboxamide]penicillanic acid.

Using 48 g of compound 12 as under Example 4 and proceeding as describedunder Example 8, 21.5 g of amorphous 6(1-amino--1 cyclohexylcarboxamidepenicillanic acid are obtained (yield: 64 percent) having the followingcharacteristics:

lodometric titer 88% Hydroxamic titer 90.3% H O (Karl Fischer) 3.8%

EXAMPLE 1 1 Procedure for the preparation of 6-(l-amino-1-cyclopentyl-carboxamido) penicillanic acid.

Using 47 g of compound 13 as described under Example 4 and proceeding asdescribed under Example 8, 19.6 g of6-(1-amino-1-cyclopentyl-carboxamido penicillanic acid are obtained(yield: 58 percent) showing the following characteristics:

lodometric titer 91% Hydroxamic titer 87% 11 0 (Karl Fischer) 5.2%

EXAMPLE 12 Procedure for the preparation of 6-[()-a-amino(3-thienyl)-acetamido] penicillanic acid.

49.5 g of compound 13 as described under Example 4 are dissolved in 500ml of water, and to the solution are added 72 g (dry weight) of theenzymatic preparation obtained under Example 3. The pH is brought to 7.8and the mixture is kept under agitation at 38C for 8 hours, keeping thepH between 7.5 and 8 during this period. At the end of the hydrolysis,the suspension is filtered from the solids. The product is isolated fromthe filtrate with the procedure described under Example 8. 29.1 g of6[()a-amino-(3-thienyl)-acetamido] penicillanic acid are obtained(yield: 71.2 percent), having the following characteristics:

Hydroxamic titer 91.3% lodometric titer 93.1% H,O (Karl Fischer) 13.6%

EXAMPLE 13 Procedure for the preparation of Pheneticillin.

To 1,000 ml of Aerobacter alcaligenes bacterial paste (ATCC 13529) areadded 50.5 g of sodium salt of compound 7, obtained as in Example 4. Thesuspension is kept under agitation at 38C and pH 7.7-8.0 for 12 hours.At the end of the enzymatic hydrolysis, the mixture is centrifuged toremove the cellular material. The clear solution is extracted with 200ml of methylisobutylketone. The extracted solution is layered with 250mlof methylisobutylketone, is cooled externally to 25C and acidified withpercent I-lCl to pH 2. The organic phase is separated from the aqueousphase and extracted two more times with 120 ml of methylisobutylketone.The pooled acid organic phases are washed with 100-150 ml of water anddried over Na SO The anhydrous solution is treated with 165 ml of molarsolution of potassium 2-ethyl-hexanote. The white precipitate formed isfiltered, washed with acetone and dried under vacuum at 35C. 34.6 g ofpotassium Pheneticillin are obtained (yield: 86 percent) showing thefollowing characteristics:

lodometric titer 88.3% H,O (Karl Fischer) 1.2% Microbiological titer86.2%

EXAMPLE 14 Procedure for the preparation of Methicillin.

Using 52 g of compound 9 as under Example 4, proceed as described underExample 13. At the end of the enzymatic hydrolysis, the clear solutionobtained after centrifugation is extracted at pH 2 with 350 ml ofmethylene chloride. The organic solution, washed with water and driedover Na SO is treated with 120 ml of 1.0 molar solution of sodium2-ethylhexanoate in methylisobutylketone. The white precipitateformed iscollected by filtration, washed first with methylene chloride and thenwith acetone, and dried under vacuum at 3540C. 28.7 g of sodium salt ofMethicillin are obtained (yield: 71 percent).

We claim:

1. Enzymatic process for the preparation of semisynthetic penicillins ofthe general formula:

or their non-toxic salts or esters where R represents a group selectedfrom 2 or 3 thienylmethyl, 2 or 3 thienylaminomethyl, an isoxazole groupof the formula:

(wherein R, represents a phenyl group or a 2- chlorophenyl or 2,6dichlorophenyl) and a group of the type:

wherein R represents an aryl, aryloxyl, arylmercaptyl; R represents ahydrogen atom or a (C -C alkyl group, (C -C alkoxyl, amino, (C -Cmonoalkylamino, (C -C dialkylamino, carboxyl, (C -C carboxyalkyl,carboxymethylacetoxyl, carboxybenzyl and, as a special case, R and Rtogether can belong to a C -C cycloaliphatic ring; R represents ahydrogen atom or, in the special case where R and R together form a C--C cycloaliphatic ring, R represents an amino, (C -C monoalkylamino or(C -C dialkylamino group; comprising enzymatically hydrolysing diacylpenicillins of the general formula:

wherein R has the same meaning as specified above, in the presence ofthe enzyme penicillin-acylase produced by microorganisms.

2. The process of claim 1 wherein the enzymatic hydrolysis of the abovespecified diacyl penicillins occurs in an aqueous medium at atemperature between 20C. and 50C. at an alkaline pH.

3. The process of claim 1 wherein the enzymatic hydrolysis of the abovespecified diacylpenicillins occurs in the presence of penicillin-acylaseproducing microorganisms or in the presence of cellular extracts of suchmicroorganisms containing penicillin acylase.

4. The process of claim 1 wherein the enzymatic hydrolysis ofdiacylpenicillins occurs in the presence of penicillin-acylase produedby microorganisms which are capable of attacking the amide group inposition 6 in the benzyl-penicillin molecule with the formation of atleast 20 percent of 6-amino-penicillanic acid within 24 hours at 38C.and pH 7.8.

5. The process of claim 1 performed in the presence ofpenicillin-acylase produced by microorganisms selected from the groupconsisting of molds, yeasts and bacteria selected from the groupconsisting of Alternatia, Aspergillus, Botritis, Cephalosporium,Cryptococcus, Emericellopsis, Epicoccum, Epiolermophyton, Fusarium,Mucor, Penicillum, Phoma, Trichoderma, Tricophyton, Trichosporon,Streptomyces, Aerobacter, Alcaligenes, Bordetella, Cellulomonas,Corynebacterium, Erwinia, Escherichia, Flavobacterium, Micrococcus,Proteus, Pseudomonas, Salmonella, Sarcina, Xantomonas, Torulopsis,Rhodotorula, Arthrobacter.

6. The process of claim 1 wherein the hydrolysis medium containsenzymatic material in such an amount as to be able to hydrolize at 37C.and pH 7.6 a 3 percent solution of benzylpenicillin in 10 hours to theextent of at least 35 percent.

7. The process of claim ll wherein an Escherichia coli strain, resistantto LOGO-3,000 'y/ml of phenylacetic acid, is used as source ofpenicillin-acylase.

8. The process of claim 3 wherein, in the enzymatic hydrolysis ofdiacylpenicillins, the penicillin acylase producing microorganisms areused in the form of cells harvested from culture media, acetonic powdersor as cells absorbed on inert carriers.

9. The process of claim 5 in which a microorganism chosen from the genusEscherichia is used as source of

2. The process of claim 1 wherein the enzymatic hydrolysis of the abovespecified diacyl penicillins occurs in an aqueous medium at atemperature between 20*C. and 50*C. at an alkaline pH.
 3. The process ofclaim 1 wherein the enzymatic hydrolysis of the above specifieddiacylpenicillins occurs in the presence of penicillin-acylase producingmicroorganisms or in the presence of cellular extracts of suchmicroorganisms containing penicillin acylase.
 4. The process of claim 1wherein the enzymatic hydrolysis of diacylpenicillins occurs in thepresence of penicillin-acylase produed by microorganisms which arecapable of attacking the amide group in position 6 in thebenzyl-penicillin molecule with the formation of at least 20 percent of6-amino-penicillanic acid within 24 hours at 38*C. and pH 7.8.
 5. Theprocess of claim 1 performed in the presence of penicillin-acylaseproduced by microorganisms selected from the group consisting of molds,yeasts and bacteria selected from the group consisting of Alternatia,Aspergillus, Botritis, Cephalosporium, Cryptococcus, Emericellopsis,Epicoccum, Epiolermophyton, Fusarium, Mucor, Penicillum, Phoma,Trichoderma, Tricophyton, Trichosporon, Streptomyces, Aerobacter,Alcaligenes, Bordetella, Cellulomonas, Corynebacterium, Erwinia,Escherichia, Flavobacterium, Micrococcus, Proteus, Pseudomonas,Salmonella, Sarcina, Xantomonas, Torulopsis, Rhodotorula, Arthrobacter.6. The process of claim 1 wherein the hydrolysis medium containsenzymatic material in such an amount as to be able to hydrolize at 37*C.and pH 7.6 a 3 percent solution of benzylpenicillin in 10 hours to theextent of at least 35 percent.
 7. The process of claim 1 wherein anEscherichia coli strain, resistant to 1,000-3,000 gamma /ml ofphenylacetic acid, is used as source of penicillin-acylase.
 8. Theprocess of claim 3 wherein, in the enzymatic hydrolysis ofdiacylpenicillins, the penicillin acylase producing microorganisms areused in the form of cells harvested from culture media, acetonic powdersor as cells absorbed on inert carriers.
 9. The process of claim 5 inwhich a microorganism chosen from the genus Escherichia is used assource of penicillin-acylase in the enzymatic hydrolysis ofdiacyl-penicillins.
 10. The process of claim 5 in which a microorganismchosen from the genus Aerobacter is used as source of penicillin-acylasein the enzymatic hydrolysis of diacyl-penicillins.
 11. The process ofclaim 5 wherein the penicillin-acylase producing microorganisms areresistant to 1,000-3,000 gamma /ml of phenylacetic acid.